U.S. patent number 4,668,637 [Application Number 06/619,467] was granted by the patent office on 1987-05-26 for method for detecting and dosing by erythroadsorption a biological substance.
This patent grant is currently assigned to Institut Pasteur. Invention is credited to Stratis Avrameas, Jean-Luc Guesdon.
United States Patent |
4,668,637 |
Guesdon , et al. |
May 26, 1987 |
Method for detecting and dosing by erythroadsorption a biological
substance
Abstract
A process for the detection of a biological substance
immobilized on a support including (1) incubating, after washing,
the substance immobilized on a support with the product of coupling
of a specific ligand with a ligand capable of reacting with
erythrocytes; (2) adding erythrocytes; (3) immersing the whole in a
solution of a fixing agent and (4) measuring the erythroadsorption,
after having turned the support over in order to permit the removal
of the red cells which have not reacted.
Inventors: |
Guesdon; Jean-Luc (Paris,
FR), Avrameas; Stratis (La Celle St-Cloud,
FR) |
Assignee: |
Institut Pasteur
(FR)
|
Family
ID: |
9277920 |
Appl.
No.: |
06/619,467 |
Filed: |
May 30, 1984 |
PCT
Filed: |
September 30, 1983 |
PCT No.: |
PCT/FR83/00198 |
371
Date: |
May 30, 1984 |
102(e)
Date: |
May 30, 1984 |
PCT
Pub. No.: |
WO84/01436 |
PCT
Pub. Date: |
April 12, 1984 |
Foreign Application Priority Data
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Oct 1, 1982 [FR] |
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82 16565 |
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Current U.S.
Class: |
436/504; 436/518;
436/520; 436/521; 436/528; 436/809; 436/826 |
Current CPC
Class: |
G01N
33/532 (20130101); G01N 33/54306 (20130101); G01N
33/555 (20130101); G01N 33/80 (20130101); G01N
33/586 (20130101); Y10S 436/826 (20130101); Y10S
436/809 (20130101) |
Current International
Class: |
G01N
33/543 (20060101); G01N 33/555 (20060101); G01N
33/58 (20060101); G01N 33/532 (20060101); G01N
33/554 (20060101); G01N 33/80 (20060101); G01N
033/567 (); G01N 033/543 (); G01N 033/555 (); G01N
033/556 () |
Field of
Search: |
;436/504,520,521,518,528 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0041426 |
|
May 1981 |
|
EP |
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2476320 |
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Feb 1980 |
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FR |
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8102790 |
|
Oct 1981 |
|
WO |
|
8200203 |
|
Jan 1982 |
|
WO |
|
Other References
Pereira, J. Immunol. Methods, 63(1983) 25-34. .
Lawny et al., Chemical Abstracts, 90(1979) #70358e..
|
Primary Examiner: Nucker; Christine M.
Attorney, Agent or Firm: Jones, Tullar & Cooper
Claims
We claim:
1. In a process for the qualitative and quantitative determination
of biological substance, comprising the steps of:
immobilizing the biological substance to be qualitatively and
quantitatively determined on a support;
coupling a specific ligand with a ligand capable of reacting the
erythrocytes and forming a product;
incubating the substance immobilized on the support with the
product of the coupling; and
adding erythrocytes of the incubating substance, wherein the
improvement comprises the steps of:
placing the incubating substance with the erythrocytes in contact
with a solution of a fixing agent while separating the erythrocytes
not fixed on the ligand, and
measuring the erythroadsorption as a function of the fixed
erythrocytes.
2. In the process as defined in claim 1, wherein the solution of
the fixing agent comprises a buffered solution of glutaraldehyde at
a concentration of approximately 0.1 to 0.5% by weight.
3. In the process as defined in claim 1, wherein the improvement
further comprises the step of:
turning the support over in the solution with the fixing agent for
the separation of the erythrocytes not fixed to the ligand.
4. In the process as defined in claim 3, wherein the solution of
the fixing agent comprises a buffered solution of glutaraldehyde at
a concentration of approximately 0.1 to 0.5% by weight.
5. In the process as defined in claim 1, wherein the support is
provided as flat support.
6. In the process as defined in claim 5, wherein the flat support
is provided of cellulose nitrate.
7. In the process as defined in claim 5, wherein the improvement
further comprises the steps of:
providing a receptacle;
fixing the support within the receptacle; and
saturating the receptacle with erythrocytes.
8. In the process as defined in claim 1, wherein the support is
provided as a microplate having wells.
9. In the process as defined in claim 8, wherein the improvement
further comprises the steps of:
filling the wells to overflowing with erythrocytes.
covering the microplate with a flexible plastic film;
immersing the covered microplate in a solution of the fixing
agent;
turning the microplate over in the solution and removing the
plastic film; and
allowing the erythrocytes that are not fixed to the ligand to
settle before measuring the erythroadsorption.
10. In a process for the qualitative determination of a biological
substance by erythroadsorption, comprising the steps of:
immobilizing a substance having a fixing affinity for the
biological substance to be determined on a support;
placing the biological substancce to be determined in a ligand
medium;
incubating the immobilized substance with the liquid medium
containing the biological substance to be determined to form a
reaction medium;
coupling a specific ligand with a ligand capable of reacting with
erythrocytes and forming a product;
incubating, after washing, the resultant reaction medium with the
product of the coupling; and
adding erythrocytes to the incubation of the resultant reaction
medium with the product of the coupling, wherein the specific
erythroadsorption is determined by the improvement comprising the
steps of:
placing the incubation of the resultant reaction medium with the
product of the coupling and the added erythrocytes in contact with
a solutIon of a fixing agent while separating the erythrocytes not
fixed on a ligand; and
determining the specific erythroadsorption by haemolysis or
counting.
11. In the process as defined in claim 10, wherein the solution of
the fixing agent comprises a buffered solution of glutaraldehyde at
a concentration of approximately 0.1 to 0.5% by weight.
12. A kit for use in the qualitative and quantitative determination
of a biological substance, comprising:
means containing anti-biological substance antibodies to be
determined coupled to a ligand capable of reaction with
erythrocytes;
means containing a phosphate buffer;
means containing a solution of a fixing agent intended for the
treatment of erythrocytes;
a support; and
a reference support.
Description
The present invention relates to the field of biology and more
particularly to the detection, by erythroadsorption, of a
biological substance immobilised on a support.
It relates in particular to improvements to the process for
detection and determination of a biological substance by
erythroadsorption described in patent application FR No.
80/15,293.
In this patent application FR No. 80/15,293 a process for detecting
and determining a biological substance by erythroadsorption has
already been described. This process employs, as a reagent for
determination, the product of coupling a specific ligand with a
ligand capable of reacting with erythrocytes, and erythrocytes as a
developer.
This process for detection and determination of a biological
substance by erythroadsorption consists in:
(1) immobilising on a support a substance having a fixing affinity
for the biological substance to be determined;
(2) incubating this substance with the liquid medium containing the
biological substance to be determined;
(3) incubating, after washing, the resulting reaction medium with
the product of coupling of a specific igand with a ligand capable
of reacting with erythrocytes.
(4) Adding erythrocytes; and
(5) Determining the erythrocyte adsorption.
This process is suitable for determining, as biological substances,
antigens, antibodies, haptens, hormones immunoglobulins and other
substances of biological interest.
According to the teaching of this Patent FR No.80 15,293, the
determination of erythroadsorption can be carried out in several
ways.
For example, it is possible to establish visually that the red
cells are adsorbed at the surface of the support on which the
substance having a fixing affinity for the biological substance to
be determined has been immobilised. In this case, the process makes
it possible to identify a particular biological substance in a
given biological liquid. If, on the other hand, the biological
liquid does not contain the particular biological substance, the
erythrocytes are not adsorbed and form a residue at the bottom of
the receptacle, for example wells of the microplate. The process
for determination by erythroadsorption according to this Patent FR
No. 80/15,293 also makes it possible to determine a given
biological substance quantitatively. For this purpose, the red
cells which have not reacted are removed, for example by suction
with a pipette. The adsorbed erythrocytes are then lysed, for
example with distilled water, and the substances liberated by the
red cells, for example haemoglobin or the substances introduced
artificially by the experimenter, are then determined by
spectrophotometry.
Haemoglobin can also be determined by an enzymatic reaction. It is
possible, for example, to employ one of the peroxidase substrates,
such as ortho-dianisidine, or ortho-phenylenediamine. The reading
is also carried out by spectrophotometry, at 400 nm for
orthodianisidine and 492 nm for ortho-phenylenediamine.
The quantity of substances liberated by the red cells, for example
the quantity of liberated haemoglobin, is proportional to the
quantity of the substance to be determined, which makes it possible
to obtain, for example, the estimate of an antigen or an antibody
present in the sample by referring to a standard range of
haemolysis of red cells produced under the same conditions.
The quantitative determination of erythroadsorption as defined
above therefore requires the removal of red cells which have not
reacted and the determination of the substances liberated by the
red cells or substances introduced artificially by the
experimenter.
In this process, the biological substance to be determined is
immobilised on a support by specific fixation, that is to say
through the intermediacy of a substance having a fixing affinity
for the biological substance to be determined.
It is also possible to determine by erythroadsorption, using the
same operating procedure, substances which are fixed on a support
by any other means, for example by passive adsorption or by
chemical bonding.
It has now been found that the determination of erythroadsorption
can be carried out without involving a step for determination of
substances liberated by the red cells which have been adsorbed or
substances introduced by the experimenter. This determination is
carried out in a simpler manner and permits a better quantification
than that described in Patent FR No. 80/15,293.
Accordingly, the present invention relates to a process for the
detection and/or determination, by erythroadsorption, of a
biological substance immobilised on a support, which consists in
determining the erythroadsorption after having, on the one hand,
removed the red cells which have not reacted and, on the other
hand, fixed the adsorbed red cells chemically on the immuno
adsorbent, solely by the use of a solution of a fixing agent.
In its most general form, the process of the invention consists
in:
(1) incubating, after washing, the substance immobilised on a
support with the product of coupling of a specific ligand with a
ligand capable of reacting with erythrocytes;
(2) adding erythrocytes;
(3) immersing the whole in a solution of a fixing agent,
(4) measuring the erythroadsorption, after having turned the
support over in order to permit the removal of the red cells which
have not reacted.
Using the solution of fixing agent employed in the process of the
invention, the erythrocytes adsorbed at the surface of the support
on which the biological substance to be detected has been
immobilised are fixed chemically and rapid elimination of
erythrocytes which have not reacted is possible. In this way, a
homogeneous layer of adsorbed and chemically fixed erythrocytes,
the density of which is a function of the concentration of the
substance to be detected, is formed on the support.
A "fixing agent" according to the invention is used to designate
any agent capable of fixing the erythrocytes on the support while
avoiding their haemolysis.
Fixing of living cells is often carried out in histology to enable
them to be studied. The fixing agents employed for this purpose
must be such that they permit the fixing of the said cells with a
minimum of disturbance of the cell structures (see Histochemistry,
PEARSE Churchill Livingstone, 3rd ed. 1968 and "La Cellule" (The
Cell) M. DURAND and B. FAVARD, Collection Hermann, Paris 1967).
In the present case, it is immaterial whether or not disturbance of
the cell structures occurs, but it is absolutely essential that the
fixing, when it takes place, is carried out under conditions which
avoid the haemolysis of the erythrocytes.
The majority of monofunctional or multifunctional fixing agents
employed currently in the field of histology are suitable for the
purpose of the invention, in particular aldehydes, such as
formaldehyde or glutaraldehyde, the latter being preferred.
The concentration of the fixing agent in the solution must be
sufficient to permit the chemical fixing of the erythrocytes which
have reacted, but it must not reach the concentration which would
cause a massive fixation of all the erythrocytes.
For example, it may be indicated that, when the fixing agent is
glutaraldehyde, the concentration of the solution should be between
0.1 and 0.5% by weight.
The coupling product employed as reactant in the process according
to the invention is the product of coupling of a specific ligand
with a ligand capable of reacting with erythrocytes.
In the present description, "specific ligand" designates any
soluble substance which can react specifically with the substance
having an affinity for the biological substance to be determined or
with the biological substance itself.
In the present description, "soluble substance" designates any
substance soluble in the media employed currently for the
biological reactions. Aqueous media may be involved, such as the
physiological media, or mixtures of aqueous and organic media.
Furthermore, the specific ligand employed must be such that the
product of coupling of the specific ligand with the ligand capable
of reacting with erythrocytes should be soluble in an aqueous
medium.
According to the invention, "aqueous medium" designates the aqueous
media, buffered or not, currently employed in the field of biology,
such as the phosphate buffer solutions, buffer solutions containing
a detergent, such as Tween or gelatine, bovine serum albumin,
bovine lactalbumin and other substances normally employed in such
fields.
The specific ligands corresponding to such a definition are
particularly antibodies, macromolecular antigens, haptens, hormones
and their receptors and similar substances. Among the specific
ligands mentioned above, those most widely employed are the
antibodies and antigens.
The ligand capable of reacting with erythrocytes is a substance
which contains sites for recognition of specific determinants of
the erythrocytes or substances fixed on erythrocytes.
It is possible to mention, as such ligands, the anti-red cell
antibodies, avidin, biotin and similar products. It is also
possible to use a lectin. Thus, the product of coupling of a
specific ligand with a ligand capable of reacting with erythrocytes
can be the product of coupling between a lectin and a specific
ligand, such as described in Patent FR No. 80/11,470, cited as a
reference. The product of coupling of a specific ligand with a
ligand capable of reacting with erythrocytes can also be the
product of coupling between an albumin and a specific ligand, such
as described in Patent Application FR No. 82/04,247, cited as a
reference.
The determination of the erythroadsorption can be carried out
either visually, or with a photometer set at 414 nm, a binocular
magnifier or an inverted microscope.
The process of the invention is suitable for detecting, either
quantitatively or qualitatively, any substance immobilised in any
way on a support.
The support employed may be any insoluble flat support, which may
or may not incorporate cavities, such as for example sheets or
strips, microplates or tubes.
The constituent substance used to make such supports may be
cellulose and its derivatives, polyacrylamide, the alkyl
polymethacrylates and other polymers of natural or synthetic
origin, and glass.
Microplates are advantageously employed to immobilise the
biological substance to be determined, for example microplates made
of polystyrene, having U-shaped or V-shaped wells or flat-bottomed
wells. It is also possible to employ individual tubes and flat
supports, for example sheets of cellulose nitrate.
For example, when the support is a sheet of cellulose nitrate, the
substance to be detected can be fixed or deposited on it directly
or indirectly, for example by impression after an electrophoresis.
In this way, human IgG can be detected.
The process of the invention is also suitable for the detection of
the clones of hybridomas which synthesize antibodies specific for
the antigens which have served to produce the said hybridomas. In
this case, the clone supernatants are removed, the supernatants are
fixed on cellulose nitrate in the form of a sheet and the presence
of the antibody is revealed by adding a specific antigen labelled
with a ligand capable of reacting with the erythrocytes.
According to the process of the invention, it is possible to detect
an isolated substance as well as a substance present in an impure
medium, given that a specific reagent for the substance to be
determined is employed.
According to another variant of the process of the invention, the
biological substance to be detected can be immobilised on the
support in a specific manner, that is to say through the
intermediacy of a substance having a fixing affinity for the
biological substance in question.
In this particular case, a substance having a fixing affinity for
the biological substance to be determined is first immobilised on
the support, then this substance immobilised in this way is
incubated with the liquid medium containing the biological
substance to be determined; it is then possible to carry out a
quantitative determination or a determination. In this form, the
process of the invention relates to an improvement to the process
of detection and determination by erythroadsorption of a biological
substance described in Patent Application FR No. 80/15,293.
According to this variant, the process of the invention consists
in:
(1) immobilising on a support a substance having a fixing affinity
for the biological substance to be estimated;
(2) incubating this substance with the liquid medium containing the
biological substance to be determined:
(3) incubating, after washing, the resulting reaction medium with
the product of coupling of a specific ligand with a ligand capable
of reacting with erythrocytes;
(4) adding erythrocytes;
(5) immersing the whole in a solution of a fixing agent, and
(6) measuring the erythroadsorption, after having turned the
support over in order to permit the removal of the red cells which
have not reacted.
The substance having a fixing affinity towards the biological
substance to be determined can be any substance capable of being
fixed in a specific manner with the said biological substance. For
example, if the biological substance to be determined is an
antibody, the substance having a fixing affinity will then be an
antigen and vice versa.
The substance having a fixing affinity for the biological substance
to be determined is immobilised on any support by the use of
conventional techniques.
The supports, for example those consisting of sheets of cellulose
nitrate on which the substance having a fixing affinity for the
biological substance to be determined is fixed, form a means of
making use of the process of the invention.
The first stages of the above process, namely stages 1 to 3, are
carried out in accordance with Patent FR No. 80/15,293. If need be,
those skilled in the art can refer to the description of this
patent.
Nevertheless, the general procedure for determining antibodies will
be recalled below, without, however, limiting the scope of the
invention to this type of determination alone.
During the first stage of the process of the invention, antigens
(Ag) are immobilised on a support.
The immobilisation of the antigens, which in this particular case
form the substance having an affinity for the biological substance
to be determined, namely the antibody, is carried out for example
by passive adsorption or, if necessary, by covalent bonding,
depending on the nature of the support.
Stage (2) consists of an incubation of the immobilised antigen with
the biological liquid containing the antibody (Ac) to be
determined, for example the serum to be titrated. After this
incubation stage, during which the antigen (Ag) interacts with the
corresponding antibody (Ac), if it is present in the serum to be
tested, the support is washed with a buffer solution, for example a
solution of a phosphate buffer, if necessary containing a detergent
such as "Tween" referred to below as PBS or PBS-Tween.
Stage (3) of the process of the invention consists in incubating
the resulting support from stage (2) with a product of coupling of
a specific ligand with a ligand capable of reacting with
erythrocytes. In this particular case, the specific ligand is an
antibody directed against the immunoglobulins of the human or
animal species in the serum to be titrated. After this incubation,
the resulting system is washed as described above to remove the
product of coupling which has not reacted. Next, erythrocytes are
added, which are adsorbed by the product of coupling only if the
serum to be titrated contains the antibody corresponding to the
immobilised antigen, otherwise the erythrocytes are not fixed and
fall to the bottom of the receptacle.
According to a preferred embodiment of the process of the
invention, whatever the manner of immobilising the biological
substance to be determined, sufficient erythrocytes are added to
fill to overflowing the wells of the microplate employed as a
support, in order to avoid the formation of air bubbles in the
wells of the said plate. The said plate is then covered, for
example with a flexible film of a plastic. Thus covered, the
microplate is immersed in the solution of a fixing agent, for
example a solution of glutaraldehyde, and is then turned upside
down. The plate floats to the surface of the solution and the film
is withdrawn. By proceeding in this manner, the red cells which are
not specifically adsorbed are removed from the plate, because they
fall to the bottom of the receptacle containing the solution of the
fixing agent.
The action of the fixing agent, such as glutaraldehyde, has two
effects:
(1) the red cells treated with glutaraldehyde settle more quickly
than the untreated red cells;
(2) the red cells bound in a specific manner to the solid phase are
then fixed chemically by glutaraldehyde, with the result that the
stability is increased.
When the unbound red cells are removed, the plate is turned over,
care being taken to avoid the entry of air bubbles into the wells.
The wells which received a negative sample are empty. The bottoms
of the wells which received the positive sample are covered with a
layer of red cells the density of which is a function of the
concentration of the substance to be determined.
The final result can be read with the naked eye, with a photometer
set at 414 nm or by means of a binocular magnifier or an inverted
microscope.
When a flat support is employed for immobilising the biological
substance to be detected, for example a sheet, it is fixed in the
bottom of a receptacle before the addition of erythrocytes and the
procedure is as above.
In order to make use of the process of the invention, use is made
of a suspension of erythrocytes the concentration of which is not
of critical importance. In practice it is sufficient to choose a
concentration capable of providing optimum erythroadsorption. By
virtue of the operation of turning the support over, which forms an
essential feature of the process of the invention, any excess
erythrocytes which may be present do not interfere with the reading
to the slightest degree, since they are removed. In general,
concentrations of the order of 0.5% are suitable. It is possible,
however, without disadvantage, to employ concentrations of 1 or 2%.
It will be noted that below 0.5% there is no certainty that the
erythroadsorption is at optimum. This represents a considerable
advantage, because there is no need to standardise accurately the
value of the concentrations of erythrocyte suspensions.
The process of the invention makes it possible to extend the use of
the erythroadsorption technique to immuno-chemical methods which
require the use of a visualising process. Thus, the processes of
autoradiography, of enzymatic colouring, and of fluorescence can
advantageously be replaced by this new process which consists in
visualising a substance by using in succession a conjugate produced
by coupling the specific ligand of the sought substance with a
lectin or an anti-red cell antibody, and a suspension of red
cells.
The present invention also relates to a kit for the detection of a
biological substance, the said kit comprising:
anti-biological substance antibodies to be determined, coupled to a
ligand capable of reacting with erythrocytes;
PBS buffer;
a solution of a fixing agent;
a support; and
a reference support.
The reference support employed in the kit according to the
invention is obtained by the detection process of the invention. To
produce this reference support, a given biological substance is
immobilised with the product of coupling of a specific ligand and
of a ligand capable of reacting with erythrocytes, erythrocytes
which are preferably fresh are then added and the whole is immersed
in a solution of a fixing agent, the support is turned over to
permit the removal of red cells which have not reacted and a
reference support is thus obtained, that is to say a support on
which erythrocytes are fixed in a quantity corresponding to the
given biological substance.
To illustrate the improvement according to the invention, but
without restricting it in any way, the following examples are
given:
EXAMPLE 1
Qualitative Detection of Human IgG Adsorbed on a Sheet of Cellulose
Nitrate
Two .mu.l of a 0.1 M borate buffer, pH 8.0, containing various
concentrations of human IgG (from 100 .mu.g/ml to 1 .mu.g/ml) were
deposited on a sheet of cellulose nitrate. After evaporation (15
minutes at room temperature) and saturation with gelatin, the sheet
of cellulose nitrate was incubated with a conjugate produced by
coupling anti-human IgG antibodies with anti-red cell antibodies. 2
hours later the sheet was washed and attached physically to the
bottom of a receptacle. The receptacle was then filled with a
suspension (0.5%) of sheep red cells. After 1 hour the red cells
were removed by gently turning the receptacle over in a buffered
physiological solution containing 0.2% of glutaraldehyde. When all
the red cells were removed the sheet was turned over. In this way,
it was possible to see a deposit of red cells when the quantity of
human IgG fixed on the cellulose nitrate sheet was equal to or
greater than 2 ng.
EXAMPLE 2
Determination of Human IgE
By virtue of this process for removal of erythrocytes which have
not reacted it is possible to establish standardisation curves by
employing given quantities of antibodies and antigens, such as
those shown in the attached figure, in which the abscissae show the
concentration of IgE in IU/ml of known solutions of IgE and the
ordinates the percentage of erythroadsorption relative to the 100%
plateau defined for an IgE concentration of 100 IU/ml.
The following procedure was followed to establish this curve: the
wells of a flat-bottomed plate were filled with 100 .mu.l of
anti-IgE antibodies (1 g/ml). The plate was incubated for 2 hours
at 37.degree. C. and one night at 4.degree. C., then washed with
PBS containing 0.1% of Tween 20. The wells then received 100 .mu.l
of a reference serum dilution containing a known quantity of IgE.
The plate was then incubated for 4 hours at 37.degree. C. and 1
night at 4.degree. C. After the incubation the plate was again
washed and each well received 100 .mu.l of a solution of anti-IgE
antibodies coupled with anti-sheep red cell antibodies. After the
incubation (2 hours at 37.degree. C.) and washing of the plate, the
wells were filled with 400 .mu.l of a suspension of sheep red cells
(0.5%). An hour later the red cells not adsorbed in a specific
manner were removed according to the procedure described in Example
1 and the absorption of the light at 414 nm was measured with a
Titertek Multiskan MC photometer. The wells which received the IgE
solution at a concentration of 100 IU/ml served as reference (100%
of erythroadsorption) to calculate the percentage of
erythroadsorption.
EXAMPLE 3
Using a strip of cellulose nitrate prepared according to the
process described in Example 1 it is possible to determine the IgG
which are present in a sample of serum. The intensity of the stains
of erythrocytes which have reacted with the anti-IgG-lectin
antibodies is compared to those present on the reference strip and
permits the detection and the determination of the IgG which are
present in the said sample.
* * * * *